Blood coagulation protein antagonists and uses therefor

ABSTRACT

Coagulation protein antagonists are disclosed, which include monoclonal-type antibodies and related cell lines disclosed for the production of specific, neutralizing antibodies against factors VII and VIIa and the tissue factor/factor VIIa bimolecular complex, which antibodies are useful for the prevention or treatment of thrombotic and related diseases, for immunoaffinity isolation and purification of factors VII and VIIa and the tissue factor/factor VIIa complex, and for determination of factors VII or VIIa and the tissue factor/factors VII or VIIa complex in a biological sample.

This application is a continuation of application Ser. No. 08/453,047,filed May 23, 1995, (now U.S. Pat. No. 5,843,422) which is acontinuation of application Ser. No. 08/163,576, filed Dec. 6, 1993,(now abandoned) which is a file wrapper continuation of application Ser.No. 07/601,454, filed Oct. 22, 1990 (now abandoned).

FIELD OF THE INVENTION

The present invention relates to antibodies and functional fragmentsthereof and, more particularly, to the treatment of patients forthrombotic disease or the prevention of thrombotic disease usinganti-thrombotic agents including immunoglobulin protein and proteinfragments or derivatives directed to certain blood coagulation-relatedproteinaceous antigens and epitopic regions thereof.

BACKGROUND OF THE INVENTION

Hemostasis is a naturally occurring process which results in thespontaneous arrest of bleeding from damaged blood vessels. For example,precapillary vessels will contract immediately when an individual iscut. Within seconds after such a cut, the process of hemostasis begins.At a site of injury with disruption of a blood vessel or exposure ofsubendothelial vascular tissue, two events rapidly occur. The two limbsof the hemostatic system, each comprised of many molecules, areactivated. The coagulation (clotting) system is immediately initiatedproducing thrombin; and blood platelets adhere to matrix proteins. Theplatelets are activated, in part by thrombin, and release adenosinediphosphate (“ADP”) leading to aggregation of additional platelets intoa growing platelet plug in concert with the conversion of fibrinogen inthe blood to the insoluble fibrin gel. This hemostatic plug isstrengthened by additional enzymatic cross-linking. Over time it isdissolved during tissue repair to result in normal tissue and bloodvessel, with or without residual pathology of the local vessel wall ortissue.

Thrombogenesis is an altered, pathogenic state of one or both limbs ofthe hemostatic system. In such states, an intravascular (arterial orvenous) thrombus results from a pathological disturbance of hemostasis.A platelet-rich thrombus, for example, is thought to be initiated by theadhesion of circulating platelets to the wall of an arterial vessel.This initial adhesion, activation by thrombin or other agonists, and theconcomitant release of ADP from platelets, is followed byplatelet-platelet interaction or aggregation. Fibrin formation isassociated with the platelet thrombus but is a minor component. Thearterial thrombus can grow to occlusive proportions in areas of slowerblood flow.

In contrast, fibrin-predominant thrombi develop initially in areas ofstasis or slow blood flow in blood vessels and may resemble a blood clotformed in vitro. The bulk of venous thrombi comprise a fibrin networkenmeshed with red blood cells and platelets. A venous thrombus canestablish a “tail” that can detach and result in embolization of thepulmonary arteries. Thus, it will be understood that arterial thrombicause serious disease by local ischemia, whereas venous thrombi do soprimarily by distant embolization.

A platelet plug formed solely by ADP-stimulating platelet interaction isunstable. Immediately after the initial aggregation and viscousmetamorphosis of platelets, as noted above, fibrin becomes a constituentof a platelet-rich thrombus. Production of thrombin occurs by activationof the reactions of blood coagulation at the site of the platelet mass.This thrombin may activate the initial adherent platelets and stimulatesfurther platelet aggregations. Platelet aggregation is stimulated notonly by inducing the release of ADP from the platelets, but also bystimulating the synthesis of prostaglandins, which as aggregatingagents-are more powerful than ADP, and by the assembly of theprothrombinase complex on the activated platelets to accelerate theformation of more thrombin, the very powerful activator of platelets.

The coagulation of blood results in the formation of fibrin. It involvesthe interaction of more than a dozen proteins in a cascading series ofproteolytic reactions. At each step a clotting factor zymogen undergoeslimited proteolysis and itself becomes an active protease. Thisclotting-factor enzyme activates the next clotting factor zymogen untilthrombin is formed which connects fibrinogen to the insoluble fibrinclot. The blood clotting factors include factor I (fibrinogen), factorII (prothrombin), tissue factor (formerly known as factor III), factorIV (Ca²⁺), factor V (labile factors), factor VII (proconvertin), factorVIII (antihemophilic globulin, or “AHG”), factor IX (Christmas factor),factor X (Stuart factor), factor XI (plasma thromboplastin antecedent,or “PTA”), factor XII (Hageman factor), factor XIII (fibrin-stabilizingfactor), and factors HMW-K (high-molecular-weight kininogen, orFitzgerald factor), PRE-K (prekallikrein, or Fletcher factor), Ka(kallikrein), and PL (phospholipid).

Fibrinogen is a substrate for the enzyme thrombin (factor IIa), aprotease that is formed during the coagulation process by the activationof a circulating zymogen, prothrombin (factor II). Prothrombin isconverted to the active enzyme thrombin by activated factor X in thepresence of activated factor V, Ca²⁺, and phospholipid.

Two separate pathways, called the “intrinsic” and “extrinsic” systems,lead to the formation of activated factor X. In the intrinsic system,all the protein factors necessary for coagulation are present in thecirculating blood. In the extrinsic system, tissue factor, which is notpresent in the circulating blood, is expressed on damaged endothelium,on activated monocytes by cells in the arteriosclerotic plaque or bycells outside the vessel wall. Tissue factor then acts as the receptorand essential cofactor for the binding of factor VII resulting in abimolecular enzyme [tissue factor:VIIa] to initiate the extrinsicpathway of coagulation. This mechanism also activates the intrinsicpathway of coagulation. The tissue factor pathway can very rapidly clotblood.

Blood can also be clotted by the contact system via the intrinsicpathway of coagulation. The mechanism is somewhat slower than the tissuefactor pathways, presumably because of the larger number of reactionsthat are required. Both the intrinsic system and extrinsic systempathways must be intact for adequate hemostasis. See Zwaal, R. F. A.,and Hemker, H. C. “Blood cell membranes and hemostasis.” Haemostasis,11:12-39 (1982).

Thrombosis and a variety of related forms of diseases are associatedwith, and result from, activation of one or more of the coagulationprotease cascades pathways, and disorders of regulation of the combinedcoagulation/anticoagulation/fibrinolytic pathways. These diseases affectapproximately 2.5 million individuals annually in the United States.Some three percent of the U.S. population over the age of 45 developsome form of thrombotic disease or disseminated coagulation each year.Other thrombotic diseases are hereditary and may affect 100,000 peopleannually. Seventy percent of such diseases are fatal by 45 years of age.

Of acquired thrombotic diseases, coronary thrombosis at about 1.5million cases per year, pulmonary thromboembolism at about 400,000 casesper year and severe septic shock at more than 300,000 cases per year,disseminated intravascular coagulation (DIC) at about 350,000 cases peryear, and deep vein thrombosis at about 175,000 cases per year,predominate. However, diseases such as menigococemia, hemorrhagic fevervirus infections, and a variety of other diseases produce significantmorbidity and mortality as well. See, e.g., Kaplan, K. “CoagulationProteins in Thrombosis.” In Hemostasis and Thrombosis, Colman, R. W., etal. eds., pages 1098 et seq. (2d Ed. J. B. Lippincott Co. 1987). Some ofthe most acutely severe forms of disseminated intravascular coagulationaffect children secondary to a variety of infectious diseases. Currenttreatment for thromboembolic disease is by no means satisfactory, andincludes the use of anticoagulants, antithrombotic drugs andthrombolytic agents.

One of the most well-known anticoagulants is heparin. Discovered in1922, heparin is a heterogenous group of straight-chain anionicmucopolysaccharides, called glycosaminoglycans, of molecular weightsthat average 15,000 daltons. Commercial heparin typically consists ofpolymers of two repeating disaccharide units: D-glucosamine-L-iduronicacid and D-glucosamine-D-glucuronic acid. It is typically prepared fromboth bovine lung and porcine intestinal mucosa, and has also beenobtained from sheep and whales.

While heparin occurs intracellularly in mammalian tissues that containmast cells, it is limited to a macromolecular form of at least 750,000daltons. Furthermore, this heparin has only 10-20% of the anticoagulantactivity of commercial heparin. Heparan sulfate, a compound similar toheparin but with less anticoagulant activity is a ubiquitous componentat the mammalian cell surface. When native heparin is released from itsbound and inactive state in the metachromatic granules of mast cells, itis ingested and rapidly destroyed by macrophages. Heparin cannot bedetected in the circulating blood.

When injected intravenously, commercially prepared heparin impairs bloodcoagulation. It acts by complexing with antithrombin III, a serineprotease inhibitor that neutralizes several activated clotting factors,ie., factors XIIa, kallikrein (activated Fletcher factor), XIa, IX, Xaand thrombin (IIa). However, it is most active in inhibiting freethrombin and activated factor X (Xa). Although antithrombin III wasthought to be the only macromolecule able to inactivate thrombin, otherplasma proteins are now known to possess this activity. Antithrombin IIIcan form irreversible complexes with serine proteases, and, as a result,the above protein factors are inactivated. Griffith, M. J.“Heparin-Catalyzed Inhibitors/Protease Reactions: Kinetic Evidence for aCommon Mechanism of Action of Heparin,” Proc. Natl. Acad. Sci. USA,80:5460-5464 (1983). Heparin markedly accelerates the velocity, althoughnot the extent of this reaction. A ternary complex is apparently formedbetween heparin, antithrombin III, and the clotting factors. Bjork, I.,and Lindahl, U. “Mechanism of the Anticoagulant Action of Heparin” Mol.Cell. Biochem., 48:161-182 (1982). Low concentrations of heparinincrease the activity of antithrombin III, particularly against factorXa and thrombin and this forms the basis for the administration of lowdoses of heparin as a therapeutic regimen.

While purified commercial preparations of heparin are relativelynon-toxic, a chief complication of therapy with heparin is hemorrhage.Heparin also causes transient mild thrombocytopenia in about 25% of thepatients, severe thrombocytopenia in a few, and occasional arterialthrombi. The mild reactions result from heparin-induced plateletaggregation, while severe thrombocytopenia follows the formation ofheparin-dependent antiplatelet antibodies complexes. It is to beunderstood that, in all patients given heparin, platelet counts must bemonitored frequently, any new thrombi might be the result of the heparintherapy, thrombocytopenia sufficient to cause hemorrhage should beconsidered to be heparin-induced, and that thrombosis thought to resultfrom heparin should be treated by discontinuation and substitution of anagent that inhibits platelet aggregation and/or an oral anticoagulant.

Severe thrombocytopenia, hemorrhage, and death have occurred even inpatients receiving “low-dose” heparin therapy. Heparin therapy is,furthermore, contraindicated in patients who consume large amounts ofethanol, who are sensitive to the drug, who are actively bleeding, orwho have hemophilia, purpura, thrombocytopenia, intracranial hemorrhage,bacterial endocarditis, active tuberculosis, increased capillarypermeability, all sorts of lesions of the gastrointestinal tract, severehypertension, threatened abortion, or visceral carcinoma. Furthermore,heparin is to be withheld during and after surgery of the brain, eye, orspinal cord, and is not to be administered to patients undergoing lumbarpuncture or regional anesthetic block. Goodman and Gillman's ThePharmacological Basis of Therapeutics, pages 1339-1344 (7th ed. 1985).

There are a number of oral anticoagulants that are also available forclinical use. Many anticoagulant drugs have been synthesized asderivatives of 4-hydroxycoumarin or of the related compound,idan-1,3-dione. The essential chemical characteristics of the coumarinderivatives for anticoagulant activity are an intact 4-hydroxycoumarinresidue with a carbon constituent at the 3 position. There are a numberof differences in the pharmacokinetic properties and toxicities of theseagents, however, and racemic warfarin sodium is the most widely usedoral anticoagulant in the United States.

The major pharmacological effect of oral anticoagulants is inhibition ofblood clotting by interference with the hepatic post translationalmodification of the vitamin K-dependent proteins among which are theclotting factors, i.e., Factors II, VII, IX and X. These drugs are oftencalled indirect anticoagulants because they act only in vivo, whereasheparin is termed a direct anticoagulant because it acts in vitro aswell. Again, hemorrhage is the main unwanted effect caused by therapywith oral anticoagulants, and such therapy must always be monitored. Inreported order of decreasing frequency, complications includeecchymoses, hematuria, uterine bleeding, melena or hematochezia,epistaxis, hematoma, gingival bleeding, hemoptysis, and hematemesis. Allof the contraindications described above in regard to the use of heparinapply to the anticoagulants as well.

Anti-platelet drugs suppress platelet function and are used primarilyfor arterial thrombotic disease, whereas anticoagulant drugs, such aswarfarin and heparin suppress the synthesis or function of clottingfactors and are used to control venous thromboembolic disorders. Thereare a number of anti-platelet drugs, the most well-known being aspirin.The efficacy of these agents for acute treatment has, however, not beenestablished and there is a real problem with aspirin hemorage.

Thrombolytic drugs include streptokinase, urokinase, tissue plasminogenactivator, and APSAC (acylated plasminogen streptokinase complex). Theseare proteins which have demonstrated efficacy for the treatment of acutethrombotic disease. They promote the dissolution of thrombi bystimulating the conversion of endogenous plasminogen to plasmin, aproteolytic enzyme that hydrolyzes fibrin. The use of these agents islimited, however, to acute thrombotic disease. Fibrinolytic agents areused primarily for the treatment of patients with established coronaryarterial thrombosis.

Effective therapy for a variety of forms of intravascular activation ofthe coagulation protease cascades, whether thrombosis or the morecatastrophic forms such as those associated with vasomotor collapse(septic shock) and other forms of disseminated intravascularcoagulations are not entirely satisfactory, and in the case of septicshock is entirely unsatisfactory. The need for effective therapy that iscapable of rapidly arresting arterial thrombogenesis is recognized as animportant therapeutic deficiency. This is evident from the recentevidence that heparin is entirely ineffective in preventing rethrombosisof the 11-20% of patients that rethrombose at the completion ofthrombolytic therapy with tissue plasminogen activator.

The present invention was made in response to these needs and relates toantagonists of factor VII and specific antagonists of the procoagulantactivity of factor VIIa and the tissue factor:factor VIIa complex. Theinvention includes monoclonal-type antibodies produced by cell systemsincluding bacteria, such as E. coli, or by hybrid cell lines,characterized in that the antibodies, or functional fragments thereof,have predetermined specificity to factor VII, to factor VIIa, and/or tothe bimolecular complex of tissue factor and factor VIIa, are effectivefor neutralization of these targets, and find application asantithrombotic agents for syndromes such as disseminated intravascularcoagulation (“DIC”) and venous thrombosis. The present invention alsorelates to the use of these monoclonal-type antibodies in methods forthe purification of factor VII, factor VIIa and the bimolecular complexreferred to above, and in methods for the immunoassay or immunodetectionof factor VII, factor VIIa and the tissue factor/factor VIIa bimolecularcomplex. The purification of factor VII, factor VIIa and the tissuefactor/factor VIIa complex from a biological sample containing theseantigens can be carried out by immuno-affinity chromatography in whichthe biological sample is passed through an immunoadsorbant column orslurry comprising the novel monoclonal-type antibodies or antibodyfragments of this invention bound to a solid base support to therebyselectively adsorb said antigenic targets. The immunoassay of factorVII, factor VIIa and the tissue factor/factor VIIa bimolecular complexfor determining the presence or concentration of these target antigensin a biological sample containing them can be carried out by contactingsaid sample with a known amount of the novel monoclonal-type antibody ofthis invention and measuring the resulting adsorbed monoclonal antibody.

Factor VII is a vitamin K-dependent, zymogen of the active serineprotease VIIa. Factor VII functions to form a complex with tissue factorin blood, and on conversion to VIIa forms the complex which thenactivates factor X by converting factor X to factor Xa. Procoagulantactivity is only associated with the tissue factor:VIIa complex. Freefactor VII and free factor VIIa, as well as the tissue factor:factor VIIcomplex, do not possess procoagulant activity. Factor VII is a singlepolypeptide chain of about 50,000 daltons that can, in a purifiedsystem, be activated by proteolytic cleavage of disulfide bonds byfactor Xa, factor IXa, thrombin and factor XIIa. Takase, T. et al.,“Monoclonal Antibodies to Human Factor VII: A One Step ImmunoradiometricAssay for VIIag, J. Clin. Pathol., 41:337-341 (1988). Human factor VII,when partially or completely activated, yields a protein comprised oftwo polypeptide chains linked by disulfide bridges. Factor VII and VIIamay be used interchangeably in this document and will be designatedVII/VIIa when target interchangeability is to be indicated.

With the advent of hybridoma technology first developed by Kohler andMilstein, it is now possible to attempt to generate monoclonalantibodies which are essentially homogenous compositions having uniformaffinity for a particular binding site. The production of mousehybridomas by these investigators is described in Nature, 256:495-497(1975) and Eur. J. Immunol., 6:511-519 (1976). Further procedures aredescribed in Harlow, E., and Lane D., “Antibodies: A Laboratory Manual”(Cold Spring Harbor Laboratory 1988). According to the hybridoma method,tissue-culture adapted mouse myelomas cells are fused to spleen cellsfrom immunized mice to obtain the hybrid cells, called “hybridomas,”that produce large amounts of a single antibody molecule. Generally,animals are injected with an antigen preparation, and if an appropriatehumoral response has appeared in the immunized animal, an appropriatescreening procedure is developed. The sera from test bleeds of theimmunized animal are used to develop and validate the screeningprocedure, and after an effective screen has been established, theactual production of hybridomas is begun. Several days prior to thefusion, which is generally carried out in the presence of polyethyleneglycol as described by Galfe et al. Nature, 266:550-552 (1977), followedby selection in HAT medium (hypoxanthine, aminopterin and thymidine) asdescribed by Littlefield, Science, 145:709-710 (1964), animals areboosted with a sample of the antigen preparation. For the fusion,antibody secreting cells are prepared from the immunized animal, mixedwith the myeloma cells, and fused. After the fusion, cells are dilutedin selective medium and plated in multi-welled culture dishes.Hybridomas may be ready to test as soon as about one week after thefusion, but this is not certain. Cells from positive wells are grown,subcloned, and then single-cells are cloned.

It is understood that hybridoma production seldom takes less than twomonths from start to finish, and can take well over a year. Theproduction of monoclonal antibodies has been described in three stages:(1) immunizing animals (2) developing the screening procedure and (3)producing hybridomas. It is also understood that any one of these stagesmight proceed very quickly but that all have inherent problems. Forexample, while immunization can be carried out with virtually anyforeign antigen of interest, many difficulties arise and variations maybe required for any specific case in order to generate the desiredmonoclonal antibodies. Prior to attempting to prepare a given hybridoma,there is no assurance that the desired hybridoma will be obtained, thatit will produce antibody if obtained, or that the antibody so producedwill have the desired specificity or characteristics. Harlow, E., andLane, D., supra at Chapter 6.

The production of monoclonal antibodies to human factor VII has beenreported, and these reagents are said to have been used to makeimmunodepleted plasma or to detect oft actor VII cross reactive materialin factor VII deficient patients. The production of monoclonalantibodies to factor VII for their use in a one step, immunoradiometricassay for factor VII:ag has also been reported. Id. The authors reportedthe preparation of three mouse monoclonal antibodies, two of which weresaid to bind, to factor VII:ag, and two of which were said to beinhibitors of factor VII in vitro. See also Howard et al., J. Clin.Chem., 35:1161 (1989). No monoclonal antibodies against either factorVII or factor VIIa have been described which therapeutically interferewith the binding of factor VIIa to tissue factor or which neutralize theactivity of the tissue factor/factor VIIa complex.

BRIEF SUMMARY OF THE INVENTION

In accordance with the present invention novel monoclonal-typeantibodies or antibody fragments are provided which can be produced byrecombinant cell lines or by hybrid cell lines, the antibodies beingcharacterized in that they have certain predetermined specificity toparticular targets, ie., factor VII, factor VIIa, the bimolecularcomplex of tissue factor and factor VIIa, and to particular epitopicregions thereof, and have neutralizing capability when combined withthese targets. By virtue of their binding to factors VII and VIIa ascompetitive, non-functional surrogates of tissue factor, they serve asantagonists to neutralize the functional activation of the coagulationprotease cascades. These antibodies are useful in the prevention andtherapeutic treatment of thrombotic conditions and related diseases inwhich the activation, of the above coagulation protease cascades plays asignificant pathogenic role. Particular antibodies are also useful inmethods for the purification of factors VII and VIIa and the tissuefactor/factor VIIa bimolecular complex, and in the immunoassay of thesetarget antigens.

Accordingly, the present invention also provides a method of preventingor treating a mammalian species for an incipient or existing thromboticdisease condition that would be alleviated by an agent that selectivelyinterferes with the extrinsic coagulation cascade, which comprisesadministering to a mammalian species in need of such treatment aprophylactically or therapeutically effective amount of a tissuefactor:factor VIIa complex antagonist. The present invention providesfor the prevention or treatment of thrombotic disease conditionsincluding acute disseminated intravascular coagulation, septic shock,coronary thrombosis, organ transplant rejection, and deep veinthrombosis. Effective tissue factor:factor VIIa complex antagonistsinclude monoclonal-type antibodies, preferably monoclonal antibodies orfragments thereof, having the tissue factor:factor VIIa complexantagonist characteristics of antibodies produced by hybridoma cell lineATCC HB 10558. The invention further provides for monoclonal antibodieshaving the ability to complex with all or some portion of a loop regionon the factor VII/VIIa molecule, preferably the structural loop regionwhich comprises the amino acids 195-208 on the factor VII/VIIa molecule.

The invention also provides for compositions useful in the prevention ortreatment of a thrombotic disease condition which comprises an effectiveamount of a tissue factor:factor VIIa complex antagonist. Suchcompositions may include the monoclonal antibodies and/or monoclonalantibody fragments referenced above. The invention further provides forsubstantially purified and purified preparations of monoclonalantibodies or monoclonal antibody fragments which substantially inhibitthe procoagulant activity of the tissue factor:factor VIIa complex. Thepresent invention also provides for hybridoma cell lines which permitthe production of such monoclonal antibodies and monoclonal antibodyfragments. Methods for producing such hybridoma cell lines are alsodescribed and claimed herein that comprise immunizing an animal specieswith an immunogen comprising one or more factor VIIa structural loopregion peptides.

Methods for inhibiting the procoagulant activity of the tissuefactor:factor VIIa complex in vivo are also described and claimed, whichcomprise administering to a mammalian species a monoclonal-type antibodyor antibody fragment that specifically reacts with said complex but doesnot substantially inhibit free factor VIIa.

The factor VIIa and the tissue factor/factor VIIa bimolecular complexagainst which the monoclonal-type antibodies of this invention havespecificity can be isolated from biological samples in the methodsdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

While the specification concludes with claims particularly pointing outand distinctly claiming the subject matter regarded a s forming thepresent invention, it is believed that the invention will be betterunderstood from the following description taken in connection with theaccompanying drawings in which:

FIG. 1 is a schematic representation of the clotting cascade, dividedinto those sequences involved in surface (contact) activation, intrinsicand extrinsic activation, and the final common pathway. Solid linesindicate direct activation of precursor zymogen to enzyme; interruptedlines show paths of both positive and negative feedback. PL indicatesphospholipid.

FIG. 2 shows the ability of 12D10 monoclonal antibody and 12D10 F(ab)fragment to inhibit the clotting of recalcified human plasma in a twostage prothrombin time test.

DETAILED DESCRIPTION OF THE INVENTION

As specifically shown in FIG. 1, blood coagulation can begin when thepageman factor (XII) undergoes contact activation and becomes bound tosurfaces. This surface-bound factor XII undergoes proteolytic activationby kallikrein (Ka) in the presence of a high-molecular-weight kininogen(HMW-K). This surface activation (contact system, intrinsic pathway)appears to initiate coagulation in vitro but is not considered to be arelevant in vivo mechanism. Deficiencies in this pathway (XII,prekallikrein and HMWK) result in prolonged in vitro clotting times butdo not cause hemostatic disorders.

Factor XIIa constitutes an arm of a feedback loop and activates more Kafrom prekallikrein (Pre-K or Fletcher factor), in the presence of HMW-K.Factor XIIa in the presence of HMW-K also activates factor XI. FactorXIa in the presence of Ca²⁺ proteolytically activates factor IX to IXa.Factor VIII, factor IXa, Ca²⁺, and phospholipid micelles (PL) from bloodplatelets form a lipoprotein complex with factor X and result in factorX activation. Factor V, factor Xa, Ca²⁺, and PL also form a lipoproteincomplex with factor II or prothrombin and activate it to IIa orthrombin. In seconds, thrombin splits two small pairs of peptides offthe large fibrinogen molecule, followed by rapid noncovalent aggregationof soluble fibrin monomers. Factor XIII, activated by thrombin to XIIIa,cross-links adjacent fibrin monomers covalently to form the insolublefibrin clot.

There is considerable evidence that tissue factor initiates coagulationin the generalized Schwartzman reaction (DIC) resulting fromendotoxinemia. Kaplan, K. Coagulation Proteins in Thrombosis. In“Hemostasis and Thrombosis” (Colman, R. W., Hirsh, J., Marder, V. J.,and Salzman, E. W.; eds.) 2nd Ed. J. B. Lippincott Co., pp. 1098, 1987.Fibrin microthrombi are uniformly found in fatal DIC and thrombosis oflarge arteries and veins may be found in 40% of cases. Minna, J. D.,Robboy, S. J., Colman, R. W. Disseminated Intravascular coagulation inMan. C. C. Thomas, 1974. Leukocytes are required participants and areinduced by endotoxin to a procoagulant (thrombogenic) state, Semararo,N., et al., “Role of leukocyte procoagulant activity inendotoxin-induced DIC: Evidence from comparative studies in rats andrabbits.” Agents Actions 11:646, 1981, 26, expressing tissue factor.Colucci, M., “Cultured human endothelial cells generate tissue factor inresponse to endotoxin.” J. Clin. Invest. 73:1893, 1983. At the same timeendothelial cells are also induced to express tissue factor, initiatecoagulation and to depress their anticoagulant properties. Moore, K. L.,“Endotoxin enhances tissue factor and suppresses thrombomodulinexpression of human vascular endothelium in vitro.” J. Clin. Invest.79:124-130, 1987.

The extrinsic coagulation cascade as currently envisioned starts withthe formation of the [tissue factor:VII] and [tissue factor:VIIa]complexes on the surface of tissue factor expressing cells. Tissuefactor is not normally expressed by blood cells or vascular endothelialcells, but following stimulation with LPS, TNFalpha or IL-1, endothelialcells transcribe and express this molecule. Though fewer molecules oftissue factor are expressed, factor VII is bound and rapidly convertedto factor VIIa by factor Xa feedback activation of the bound factor VII.Endothelial cell factor IX/IXa receptor (IX-R) and factor VIII(activated to factor VIIIa by Xa or thrombin feedback) markedly enhancethe kinetics of factor Xa generation by limited proteolytic activationof factor X. Cell surface associated factor V (activated by thrombinfeedback) further amplifies the Vmax of factor Xa and preventsinhibition by plasma heparin:AT-III protease inhibitor. Prothrombin isefficiently converted to thrombin to convert fibrinogen to fibrin, leadsto release of Plasminogen activator inhibitor I, serves as a chemotacticagent, aggregates platelets, activates Mac-1 receptor of monocytes, andhas other inflammatory effects.

There are no presently effective drugs for the inhibition of theextrinsic pathway. The use of heparin, shown to be without benefit, isnevertheless continued clinically with the attendant secondary problemsof its platelet effects. In DIC with depletion of antithrombin III thereis no benefit to heparin since it is not a direct anticoagulant, only acofactor for the thrombin inhibitor antithrombin III when present as a[heparin:antithrombin-III] complex. Anti-platelet drugs do not inhibitthe coagulation protease cascade, and they diminish the necessaryhemostatic properties of platelets. Warfarin therapy to interfere withVitamin K supported gamma carboxylation of factors VII, X, IX andprothrombin is too slow and is associated with reduced activity of thenatural anticoagulation pathways due to inhibition of gammacarboxylation of Protein C and Protein S. The present inventionaddresses this need by inhibiting the reaction pathway at the earliestpossible step, the initiating proteolytic complex of [tissuefactor:VIIa], which will block the intravascular initiation ofcoagulation by tissue factor positive cells, e.g., endothelial cells,monocytes, and tissue factor positive foam cells in atheroscleroticplaques.

The present invention employs a neutralizing antagonist surrogatecofactor, preferably a monoclonal antibody or antibody fragment, to thefunctional bimolecular initiation complex of tissue factor and VII/VIIaand, more preferably, to a neoantigen(s) induced on the bimoleculartissue factor:VIIa complex or alternatively, a neutralizing monoclonalantibody to VII or VIIa, preferably to a structural loop region thereof.Binding of such monoclonal antibodies to (tissue factor:VIIa) so as toblock the active site of VII/VIIa, dissociate VIIa from tissue factor orcompetitively inhibit the association of the substrate serine proteasezymogens factors X or IX will inhibit initiation of coagulation onvascular cells and arrest one of the major pathogenetic processes inthrombotic diseases.

Though the activation of coagulation has long been recognized as centraland required for thrombus formation and growth and for disseminatedintravascular coagulation, many mechanisms are put into action,particularly in the most virulent forms of septic shock. It has beendemonstrated that monoclonal antibodies to TNFalpha are capable ofprotecting baboons from endotoxin mediated septic shock, Tracey K. J.,“Anti-cachectin/TNF monoclonal antibodies prevent septic shock duringlethal bacteraemia,” Nature 330:662, 1987, since TNFalpha is induced byendotoxin, IL-1 and the toxic shock toxin 1. Michie, H. R., “Detectionof circulating tumor necrosis factor after endotoxin administration.”N.Eng. J. Med. 318:1481, 1988, Jupin, C., et al., “Protein C preventsthe coagulopathic and lethal effects of escherichia coli infusion in thebaboon.” J. Clin. Invest. 79:18, 1987. However, anti-TNFalpha oranti-LPS monoclonal antibodies may have low efficacy once the pathologicprocess has been established. Recently, it has been shown that activatedprotein C, the natural anticoagulant protein given in massive doses iscapable of arresting and even reversing early ongoing septic shock.Taylor, F. B., “Protein C prevents the coagulopathic and lethal effectsof escherichia coli infusion in the baboon.” J. Clin. Invest. 79:918,1987. Now, evidence from the same group with the same model indicatesthat arresting the initiation of coagulation with monoclonal antibody totissue factor is effective in treating septic shock in lethal challengeof baboons with E. coli (Edgington, et al., “Tissue Factor: MolecularBiology and Significance in the Pathophysiology of Gram-Negative SepticShock,” In: Microbiological, Chemotherapeutical and ImmunologicalProblems in High Risk Patients. E. Garaci, et al., Eds., Raven Press,New York, Vol. 61, pp. 29-37 (1989).

One method useful for the production of anti-protein antibodies involvesthe use of synthetic peptides from regions of the protein sequence occuron the surface of the protein to raise and/or screen for desirableantibodies. In the case of Factor VIIa however, there is no availableexperimental data on the structure. Only the amino acid sequence isknown. Factor VIIa has some sequence and structural homology in itscatalytic domain to several other proteases whose structures have beendetermined by X-ray crystallography. The sequences of these proteaseswere analyzed and the sequence of the catalytic domain of factor VIIawas compared. Regions of the factory; VIIa molecule were discovered thatwere highly conserved in structure often representing the core structureof the protein, as well as regions that were more variable. The regionsin sequence with variable structures, herein denominated “loops,” werediscovered on the surface of the catalytic domains.

Eleven loop regions were identified in the sequence of the catalyticdomain of factor VIIa. The include peptides comprising amino acids165-177, 195-208, 209-218, 234-248, 248-258, 263-278, 285-295, 313-321,330-339, 348-360, and 367-390. A computer model of the structure of thecatalytic domain of factor VIIa was constructed and the location of thestructurally variable loops ascertained. One set of loops was discoveredto be located near the catalytic site of factor VIIa, and anotherclustered around the activation site where various proteases cleave theenzymatically inactive single-chain form, factor VII, to the activetwo-chain form, factor VIIa.

Anti-factor VII/VIIa antibody epitopes targeted for neutralization, wereused to generate antibodies subsequently determined to neutralize orinhibit the activity of Factor VIIa by binding to a loop region. Whenthese loops are near the active site, binding of these antibodies blocksaccess, for example, to the site by substrates such as Factor X andthereby inhibits the function of factor VIIa. In this manner, antibodiesare prepared that block the extrinsic coagulation pathway.

Description of hybridoma preparation and initial characterization ofmonoclonal antibodies against factor VII/VIIa and the tissuefactor/factor VIIa complex, is set forth in Example 1 below. Parametersare described relating to preparation of the antigen, dose and form ofantigen, route of inoculation and immunization protocol, hybridomapreparation, and the screening, isolation and initial characterizationof monoclonal antibodies. The properties of a monoclonal antibodydesignated 12D10 (ATCC HB 10558) are set forth and described. Theantibody was shown to bind factor VII/VIIa and to dramatically inhibitthe activity of the tissue factor:factor VIIa complex. As shown by theresults in Example 2, the 12D10 antibody was also able to inhibit theactivity of free factor VIIa. The 12D10 monoclonal antibody was shown inExample 3 to be specific to amino acids 195-208 region of the factorVII/VIIa molecule. Fragmentation of the 12D10 monoclonal antibody asdescribed in Example 5, furthermore, was beneficially shown not toaffect its clotting inhibition activity.

Antibodies, or the desired binding portions thereof including F(ab) andFv fragments, can also be generated using processes which involvecloning an immunoglobulin gene library in vivo. Huse et al., “Generationof a Large Combinatorial Library of the Immunoglobulin Repertoire inPhage Lambda,” Science 246:1275-1281 (Dec. 8, 1989). Using thesemethods, a vector system is constructed following PCR amplification ofmessenger RNA (mRNA) isolated from spleen cells with oligonucleotidesthat incorporate restriction sites into the ends of the amplifiedproduct. Separate heavy chain and light chain libraries are constructedand may be randomly combined to coexpress these molecules together andscreened for antigen binding. Single chain antibodies may also beprepared and utilized.

Additional monoclonals that neutralize the factor VIIa-tissue factorbimolecular cell surface activation complex can be made and selectedfrom three classes of antithrombotic monoclonal antibodies. Thespecificity of the three classes of antibody include those reactive withfactor VII and VIIa and neutralize amidolytic activity. Two subsets ofantibody are generated. One will inhibit factor VIIa activity bypreventing the association of tissue factor and factor VII/VIIa and theother will directly inhibit the activity of factor VIIa. The secondclass includes those monoclonals reactive with only Factor VIIa andneutralize amidolytic activity, while the third includes those reactivewith neutralizing neoepitopes expressed as the result of association oftissue factor and factor VII. These neoepitopes would not be expressedon either free tissue factor or factor VII and therefore restricted tothe coagulation initiation complex.

Each of the three classes of antibody represent further uniquemechanistic approaches for antithrombotic therapy. Antibodies in thefirst class are defined by the specificity of the 12D10 monoclonal.Antibodies in,the second specificity class are developed by immunizingmice with recombinant factor VIIa. Monoclonals that react with factorVIIa but not factor VII are selected. The desirable reagent will inhibitactivity of preformed tissue factor:factor VIIa complexes. Neoepitopesexpressed on the functional bimolecular complex will be immunogenictargets for the development of monoclonals that neutralize activity.These antibodies are prepared by performing in vitro immunization ofmurine splenocytes using preformed complexes of tissue factor:factorVIIa in an optimal environment of phospholipids. In vitro immunizationis preferred due to the proteolytically labile nature of the complex invivo; however, standard in vivo immunization of heparinized mice canalso be employed. Screening is used to identify only those antibodiesreactive with the tissue factor:factor VIIa complex, as opposed to thosethat are reactive with either free-tissue factor factor VIIa. Theseantibodies will only inhibit coagulation at the site of injury oractivation and normal hemostasis will not be compromised.

EXAMPLE 1

Preparation of hybridomas and identification of desired monoclonalantibodies was as follows. Female balb/c mice were immunized withpurified human factor VII (factor VII) isolated from pooled human plasmaover a period of approximately six months. Complete Freund's adjuvantwas used for primary immunization and incomplete Freund's adjuvant forbooster immunization. One to ten micrograms of protein was used perimmunization. Route of immunization was both intraperitoneal andsubcutaneous. Three days prior to fusion mice received an intravenousperfusion boost of purified factor VII (20 μg) in saline. Spleens wereremoved and spleen cells were fused to the SP2/0 myeloma followingstandard hybridoma methods.

The screening strategy employed a three-staged methodology. Primaryscreening identified hybridoma antibodies that reacted with factor VIIor factor VIIa antigen. Secondary screening identified antibodiescapable of inhibiting the functional activity of factor VIIa asindirectly assessed in a factor X activation chromogenic substrateassay. Tertiary screening assessed clotting inhibition of recalcifiedplasma in a two-stage prothrombin time test.

The primary screening assay was a radioimmunoassay where antibodies weretested for binding to ¹²⁵I-factor VII. Briefly, ninety-six wellpolyvinyl chloride microliter plates were passively coated with affinitypurified goat anti-mouse IgG obtained from Sigma Chemical Company, St.Louis, Mo. Antibody-coated plates were blocked with bovine albumin andculture supernatants (diluted at least 1:50) were bound to the plates.Plates were washed to remove unbound antibody and ¹²⁵1-factor VII orfactor VIIa (100,000 cpm/well; specific activity of factor VII=6 μCi/μg;specific activity factor VIIa=4 μCi/μg) was added followed byincubation. Plates were washed to remove unbound factor VII and wellswere transferred to a gamma counter to determine bound labeled factorVII. Negative controls include hybridoma culture supernatant from a cellline secreting irrelevant monoclonal antibody such as anti-tPA, sterileculture medium and buffer. Competition of binding of ¹²⁵I-factor VII toantibody with excess unlabeled ligand is used to further demonstratespecificity.

The second screening was used to evaluate the ability of isolatedantibodies to inhibit tissue factor catalyzed factor VII activity asreflected by the conversion of factor X to factor Xa. The human bladdercarcinoma cell line J-82 (ATCC HTB-1) expresses cell surface-associatedtissue factor and is used as the source of tissue factor andphospholipids. A chromogenic substrate for factor Xa is used, whichdevelops color proportional to the amount of factor VIIa activity.Conversely, color is not developed if factor VII activity is blocked.The assay is performed as follows. J-82 cells are suspended intris-buffered saline at a concentration of 1×10⁵ cells per mL. Fiftymicroliters of cell suspension is added to individual wells of a 96-wellpolystyrene microliter plate. Fifty microliters of hybridoma culturesupernatant diluted at least 1:10 is added to appropriate wells followedby 25 μL of 20 mM CaCl₂. Negative control is irrelevant hybridomaculture supernatant (such as anti-tPA supernatant) and positive controlis 1 μM PPACK (d-phenylalanine-proline-arginine-chloromethylkefone).Twenty-five microliters of 90 nM factor X and 50 μL of substrateSpectrozyme Xa are added to each well. Following thirty minuteincubation at room temperature, OD-405 is determined. Maximum activation(negative control) was obtained with samples that were treated withbuffer or irrelevant hybridoma culture supernatant. Complete inhibition(positive control) assessed with the PPACK from this assay is shown inTable 1 below.

TABLE 1 Results of Factor X activation assay Sample Treatment OD-405Buffer 1.101 Anti-tPA hybridoma 1.151 culture supernatant (1:10) PPACK(1 μM) 0.023

The properties a monoclonal antibody isolated from a preferredhybridoma, designated 12D10, in the factor VII/VIIa binding assay andthe factor X activation assay are show in Tables 2 and 3 below.

TABLE 2 Identification of Hybridoma ANtibody 12D10 in F.VII/VIIa AntigenBinding Assay Antibody ¹²⁵I-rec Antigen CPM Bound 12D10 rec* F.VII 7039712D10 rec F.VIIa 30489 12D10 rec F.VII + 50-fold molar excess cold recF.VII 1878 12D10 rec VIIa + 50-fold molar excess cold rec F.VIIa 2771anti-tPA rec F.VII 5419 anti-tPA rec F.VIIa 3734 Culture medium recF.VII 2232 Culture medium rec VIIa 4311 *rec = recombinant

Results in Table 2 show that the 12D10 monoclonal antibody reactsspecifically with both factor VII and factor VIIa antigen.

TABLE 3 Identification of Neutralizing Activity of Hybridoma antibody12D10 Inhibitor Percent Inhibition of F.X Activation Buffer 0 Anti-tPAMonoclonal Antibody 3 PPACK (1 μM) 100 12D10 (1:10 dilution) 100

Results in Table 3 indicate that the 12D10 monoclonal antibody inhibitsactivity of the tissue factor:factor VIIa complex.

EXAMPLE 2

The factor X activation assay was used to determine the mechanism ofinhibition by the 12D10 monoclonal antibody. Hybridoma culturesupernatants diluted 1:50 were preincubated with either rF.VIIa orrF.VIIa (rF.7VIIa and rF.VIIa indicates recombinant source of specifiedmolecule) precomplexed to cellular tissue factor expressed on thesurface of J-82 cells. Antibody incubations were for 30 minutes at roomtemperature. Efficiency of antibody blocking was assessed in the factorX activation assay. Controls included irrelevant hybridoma antibody(anti tPA) and a monoclonal to factor VIIa which is known to prevent theassociation of tissue factor with factor VIIa but will not inhibitactivity following complex formation (Mab 1296). The optimal specificityfor an antithrombotic monoclonal antibody is one that inhibits thecellular complex of tissue factor and factor VIIa. Results from thisexperiment are presented in Table 4.

TABLE 4 Mechanism on Inhibition of F.VIIa by Hybridoma Antibody 12D10Monoclonal Antibody Preincubation % Inhibition of F.X Activation 12D10F.VIIa 97 ± 0  12D10 TF:VIIa 98 ± 0  1296 F.VIIa 85 ± 1  1296 TF:VIIa 22± 12 anti tPA F.VIIa 0 ± 0 anti tPA TF:F.VIIa 3 ± 5

These results demonstrate the ability of monoclonal antibody 12D10 toinhibit the activity of both free factor VIIa and cellular complexes oftissue factor and factor VIIa, an important property for the disclosedtherapeutic anticoagulant.

EXAMPLE 3

Detailed specificity of the 12D10 monoclonal antibody was evaluated asfollows. A series of synthetic peptides representing linear sequencesfrom the two gla domains, EGF domains, the light chain and the catalyticsite of factor VIIa were tested for reactivity with the 12D10 monoclonalantibody. The experiment was performed by coating microtiter wells with12D10 monoclonal antibody and reacting the capture antibody with 25 μLof a 100 μM solution of indicated peptide for 30 minutes at 37° C.following this incubation, 25 μL of 1 nM¹²⁵I-factor VIIa was added toeach well for one hour at room temperature. Wells were washed and bound¹²⁵I-factor VIIa was determined. Factor VIIa peptide containing aminoacid residues 195-208 prevented the binding of the 12D10 monoclonalantibody to ¹²⁵I-factor VIIa.

This result was confirmed by direct binding of the 12D10 monoclonalantibody to the Factor VIIa peptide 195-208. Peptides were adsorbed tomicrotiter wells at a concentration of 1 mg per mL at 37° C. for 2hours. Wells were blocked with albumin and reacted with 12D10 monoclonalantibody (10 μg per mL) for 2 hours at 37° C. Goat anti-mouse IgGperoxidase conjugate was used to demonstrate bound monoclonal antibodyfollowed by substrate development and determination of OD-450. Peptidesrepresenting the gla (2 peptides), EGF (8 peptides), light chain (2peptides) and catalytic (11 peptides) domains were tested. The catalyticdomain peptide 195-208 bound 12D10 monoclonal antibody (OD-450=0.450)while all others were negative (OD-450≧0.042). The specificity of the12D10 monoclonal antibody for the catalytic domain of factor VIIa isconsistent with our discovery that the antibody binds to VIIa before andafter reaction with tissue factor:VII complex.

EXAMPLE 4

Characterization of the activity of F(ab) fragments of 12D10 antibodywas carried out as follows. The production of F(ab) fragments of the12D10 monoclonal antibody was accomplished using a commercial kit(Bioprobe International Tustin, Calif.). F(ab) fragments are prepared bypapain cleavage of IgG. Papain is inhibited and removed by addition ofanti-papain polyclonal antibody. Protein A chromatography is used toclear Fc fragments, intact IgG and immune complexes containing papain.The F(ab) fragments were further purified by size exclusionchromatography using a Superose 12 column. Monoclonal antibody 12D10,purified as described above, was analyzed before and after papaindigestion. The resulting purity of the 12D10 IgG and F(ab) fragments wasabout 95%. The activity of these F(ab) fragments was compared to intact12D10 IgG in both the factor X activation assay and clotting inhibitionassays. Analysis of 12D10 IgG and F(ab) fragments in the factor Xactivation assay was performed as described above, except that optimallyrelipidated recombinant human tissue factor was substituted for J-82cells as a source of tissue factor. This modification enhances theprecision and the reproducibility of the assay and alleviates the needto perform cell culture to obtain J-82 cells. In these experiments,factor VII and was preincubated for 30 minutes at room temperature withthe 12D10 IgG or F(ab) prior to introducing the immune complexes intothe factor X activation or clotting assays.

TABLE 5 Inhibition of F.X Activation With 12D10 IgG and F(ab) MolarRatio of Antibody Binding Inhibitor Sites to Factor VIIa PercentInhibition 12D10 IgG 100 100 12D10 IgG 10 100 12D10 IgG 1.0 100 12D10IgG 0.1 28 12D10 F(ab) 100 100 12D10 F(ab) 10 100 12D10 F(ab) 1.0 10012D10 F(ab) 0.1 45 Anti-tPA IgG 100 5 Anti-tPA IgG 10 0 Anti-tPA IgG 1.02 Anti-tPA IgG 0.1 0

These results, shown in Table 5, indicate that the fragmentation of the12D10 antibody did not result in a loss of biological activity. Activityof the IgG and F(ab) fragments are essentially identical under theseexperimental conditions. The potency of the 12D10 antibody is evidentfrom this result as inhibition at 1:1 molar ration of antibody site toenzyme is observed. Inhibition of the factor X activation assay atratios of factor VIIa:monoclonal less than 1 can be explained by thefact that the assay readout is an extreme-amplification of the residual(uninhibited) quantity of factor VIIa activity present in the sample.

EXAMPLE 5

The 12D10 monoclonal antibody will inhibit the clotting of recalcifiedhuman,plasma in a two stage prothrombin time test. The effect of theF(ab) fragmentation process on this property was analyzed as follows.Plasma was diluted 1:2 with 2 mM sodium citrate. IgG or F(ab) atindicated concentrations (50 μL) was added to 100 μL of diluted plasmaand incubation was performed at room temperature for 20 minutes. Humanthromboplastin (Thromborel S: Behring Diagnostics) was diluted 1:1000 in30 mM CaCl₂ and 200 μL was added to the plasma/antibody solution.

Clotting times were determined in a Coagamate optical coagulometer(Organon Technica). Clotting times are converted to percent tissuefactor activity by an algorithm previously described, Hvatum, M. andPrydz H. Studies on Tissue Thromboplastin. Solubilization with SodiumDioxycholate. Biochem. Biophys. Acta. 130:92-101 (1966). The results arepresented in FIG. 2. These results demonstrate that the 12D10 F(ab)fragment is a potent inhibitor of clotting of human plasma.

We claim:
 1. A monoclonal antibody which has the in vitro coagulationantagonist characteristics of an antibody produced by hybridoma cellline ATCC HB
 10558. 2. A monoclonal antibody which has the procoagulantantagonistic activity characteristics of monoclonal antibody 12D10.
 3. Asubstantially purified preparation of a monoclonal antibody which hasthe procoagulant inhibiting activity characteristics of monoclonalantibody 12D10.
 4. A monoclonal antibody having the tissue factor:factorVII/VIIa complex antagonistic properties of monoclonal antibody 12D10.5. The antibody of claim 4, wherein said properties include bindingamino acids 195-208 of a factor VII/VIIa molecule, inhibiting theactivity of the tissue factor:factor VIIa complex, and inhibiting theactivity of free factor VIIa.